CN113533552B - A method for synchronous detection of drugs that affect ARR during the detection of renin activity by liquid chromatography tandem mass spectrometry - Google Patents

Abstract

The application provides a method for synchronously detecting therapeutic drugs affecting ARR (ratio of plasma aldosterone to renin activity) in the process of detecting renin activity by liquid chromatography tandem mass spectrometry, which realizes qualitative screening of drugs affecting ARR values while detecting plasma renin activity by liquid chromatography tandem mass spectrometry, and can be used for assisting in analysis and judgment of whether ARR is negative, positive, false negative or false positive. The effective extraction of angiotensin I and 43 antihypertensive drugs is realized while the protein precipitation is carried out on the sample; the characteristics of high flux, high specificity and high sensitivity of the liquid chromatography tandem mass spectrometry technology are utilized to synchronously detect multiple indexes including angiotensin I, 43 antihypertensive drugs and the like on an extracted sample; and (3) performing primary screening of the medicine by using MRM mass spectrum parameters of the screened medicine, and rechecking according to the retention time of the medicine. ARR detection values are jointly analyzed in combination with drug screening results, false positive or false negative results of detection are effectively distinguished while stopping drug treatment of patients, accuracy of actual primary aldosteronism and clinical renin activity detection is improved, and clinical popularization and application are facilitated.

Description

Effect of drug synchronization on ARR during detection of renin activity by liquid chromatography tandem mass spectrometry detection method

This application claims the priority of an earlier Chinese application, application number: 2021106145678, with an application date of June 2, 2021; all its contents are regarded as a part of the present invention.

technical field

The present invention relates to the technical field of chemical analysis, in particular to a qualitative detection method for primary aldosteronism, in particular to the detection of the ratio ARR of plasma aldosterone concentration (PAC)/plasma renin activity (PRA) and Judgment method.

Background technique

Primary aldosteronism (PA, referred to as primary aldosteronism) is a common endocrine hypertension, which is characterized by the autonomous secretion of aldosterone by the adrenal cortex, resulting in sodium retention and potassium excretion in the body, increased blood volume, renin-angiotensin The system activity is inhibited, and the main clinical manifestations are hypertension and hypokalemia.

The ratio ARR of plasma aldosterone concentration (PAC)/plasma renin activity (PRA) is the most reliable method for screening primary aldosteronism. Plasma renin activity is an index to measure the production efficiency of angiotensin I in peripheral blood under the catalysis of renin, which is of great significance in the classification and diagnosis of hypertension caused by renin-angiotensin-aldosterone system (RAAS).

In 2010, led by the Endocrinology Society of the Chinese Medical Association, 1,656 patients with refractory hypertension were screened for PA in 19 centers in 11 provinces across the country, and the reported prevalence was 7.1%. Research published by Professor Li Qifu's team showed that the incidence of primary aldophylaxis in newly diagnosed hypertension was more than 4.0%. In the 2020 edition of "Expert Consensus on the Diagnosis and Treatment of Primary Aldosteronism", ARR is the preferred screening indicator for primary aldosteronism, and it is believed that primary aldosteronism should be tested for hypertension, especially those with refractory hypertension and newly diagnosed hypertension. The screening has practical guiding significance for clinical work, and it is recommended that all newly diagnosed hypertensive patients should be screened for primary aldehydemia.

In the expert consensus, it is further pointed out that the ARR value will be affected by many factors including age, sex, diet, medication, body position, serum potassium and creatinine, which may lead to false positive or false negative results, especially the treatment used Drugs are concerned. The consensus requires that the detection of ARR must be performed when the drugs that have a greater impact on the ARR value are stopped for at least four weeks to avoid false positive or false negative results; special attention needs to be paid to ACE inhibitors (ACE-Is), dihydropyridine calcium Channel blockers (CCBs) and angiotensin II receptor blockers (ARBs) can reduce ARR by reducing PAC and greatly increasing PRA levels, resulting in false negative test results, and the above drugs need to be stopped for at least 2 weeks Test again. However, since PA often causes severe hypertension, discontinuation of the drug is potentially harmful to the patient.

The determination of ARR includes plasma aldosterone (PAC) and plasma renin activity (PRA). At present, radioimmunoassay is widely used to measure PRA, and the rate of conversion of angiotensinogen to angiotensin Ⅰ in unit time and unit volume indirectly reflects the renal activity in plasma. level of activity. Most centers also measure blood aldosterone (PAC) by radioimmunoassay. Immunoassay for the determination of renin activity and aldosterone content has inherent poor specificity, cross-coupling reactions and other defects in immunoassays, and it is impossible to screen for hypertension treatment drugs that affect ARR values, and patients need to face the risk of drug withdrawal testing .

Therefore, there is an urgent need to find a simpler and more convenient ARR detection method, which can not only prevent patients from stopping drug treatment, but also effectively reduce the false positive or false negative results of primary aldosteronism detection, and improve the clinical practice of ARR and renin activity detection. accuracy.

Contents of the invention

Aiming at the problems existing in the prior art, the present invention provides a method for synchronous qualitative detection of antihypertensive drugs that affect ARR during the detection of renin activity by liquid chromatography tandem mass spectrometry, mainly in the detection of blood plasma by liquid chromatography tandem mass spectrometry At the same time as the renin activity, the qualitative screening of drugs that affect the ARR value is realized. The joint analysis of ARR detection value combined with interference drug screening results can effectively reduce the false positive or false negative results of the detection while preventing patients from stopping drug treatment, and improve the accuracy of detection of actual primary aldosteronism and clinical renin activity. It is convenient for clinical popularization and application.

On the one hand, the present invention provides a method for synchronous qualitative detection of antihypertensive drugs that affect ARR during the detection of renin activity by liquid chromatography tandem mass spectrometry, mainly while detecting the content of angiotensin I in plasma samples, to realize Qualitative screening of antihypertensive drugs that affect ARR in plasma, combined with the impact of positive screening drugs on ARR, combined with the concentration of aldosterone, can be used for comprehensive judgment and analysis of whether ARR may be negative, positive, false negative, or false positive kind of.

Further, there are a total of 43 kinds of hypertension treatment drugs that affect ARR, including nine kinds of β-blockers, five kinds of potassium-sparing diuretics, three kinds of potassium-sparing diuretics, and ten kinds of angiotensin converting enzyme inhibitors , six kinds of angiotensin receptor antagonists, eight kinds of calcium ion antagonists, one kind of central α2 receptor agonists and one kind of non-steroidal anti-inflammatory drugs; the calculation formula of the ARR is: ARR=aldosterone concentration /Angiotensin I production rate.

According to the 2020 edition of "Expert Consensus on the Diagnosis and Treatment of Primary Aldosteronism", the causes of ARR false positive or false negative drugs are as follows:

Note: ARR: plasma aldosterone-to-renin activity ratio; ACEI: angiotensin-converting enzyme inhibitor; ARB: angiotensin receptor antagonist; CCB: calcium ion blocker

And in the 2020 edition of "Expert Consensus on the Diagnosis and Treatment of Primary Aldosteronism", the measurement of ARR must be carried out at least four weeks after the drug that has a greater impact on the ARR value is stopped to avoid false positive or false negative results. Drugs such as ACEI, ARB, and CCB should be stopped for at least 2 weeks and tested again to avoid false negative results. However, since PA often causes severe hypertension, discontinuation of the drug is potentially harmful to the patient.

After market research, the research team of the present invention has collected 43 kinds of hypertension treatment drugs that have a greater impact on ARR and are used more frequently, and established its liquid chromatography tandem mass spectrometry analysis method, creatively in the process of renin activity detection Simultaneous detection of drugs that affect ARR avoids the risk of drug withdrawal, improves the accuracy of clinical ARR judgments, and effectively reduces false positive or false negative results in primary aldosteronism detection, which is of great clinical significance.

The plasma aldosterone concentration (PAC) in the ARR detection process involved in the present invention adopts solid-liquid extraction (SLE) to pretreat the plasma sample, and detects it with liquid chromatography tandem mass spectrometry.

Further, the hypertension treatment drugs affecting ARR are respectively:

The nine kinds of beta-blockers include arolol, atenolol, bisoprolol, esmolol, labetalol, metoprolol, nebivolol, propranolol , sotalol;

The five potassium-sparing diuretics include bumetanide, furosemide, hydrochlorothiazide, indapamide, and torasemide;

The three potassium-sparing diuretics include amiloride, eplerenone, and spironolactone;

The ten kinds of angiotensin converting enzyme inhibitors include benazepril, captopril, enalapril, fosinopril, imidapril, lisinopril, perindopril, quinapril Li, ramipril, trandolapril;

The six angiotensin receptor antagonists include candesartan, irbesartan, losartan, olmesartan, telmisartan, valsartan;

The eight kinds of calcium ion antagonists include amlodipine, benidipine, felodipine, flunarizine, lercanidipine, nicardipine, nifedipine, nimodipine;

The central α2 receptor agonist includes methyldopa;

The one non-steroidal anti-inflammatory drug includes aspirin.

Further, the β-receptor blockers, central α2 receptor agonists, and non-steroidal anti-inflammatory drugs can all increase ARR, resulting in false positive results; the potassium-sparing diuretics, potassium-sparing diuretics, Angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, and calcium ion antagonists can all reduce ARR, resulting in false-negative results.

Any one of β-receptor blockers, central α2 receptor agonists, and non-steroidal anti-inflammatory drugs can reduce the concentration of aldosterone and renin activity in patients, and the decrease in renin activity is greater than the decrease in aldosterone concentration , leading to false positive results.

Potassium-sparing diuretics or potassium-sparing diuretics can increase the concentration of aldosterone and renin activity in patients, and the increase in renin activity is greater than the increase in aldosterone concentration; angiotensin converting enzyme inhibitors, angiotensin receptor antagonists Any one of the calcium antagonists can reduce the concentration of aldosterone and increase the activity of renin in the patient, resulting in false negative results.

Further, the step of analyzing the value of ARR is: analyzing and judging ARR as one of negative, positive, false negative, or false positive through the AI guidance system for clinical medicine; the AI guidance system for clinical medicine includes a patient information module , database module and decision tree system.

Further, the information module is used to record the basic information of the patient; the database module is used to store the test results of ARR and the test results of the drug for treating hypertension; the decision tree system automatically analyzes and judges that the ARR index is negative according to the test results , positive, false negative, or false positive.

Further, when the primary aldosteronism is judged to be one of negative, positive, false negative, or false positive according to the analysis of the test results, the cut-off value of ARR is 30.

Further, the analysis and judgment method of the decision tree system is: when the detection result of ARR is a negative result slightly lower than 30, if it is also detected that the patient contains a drug that can reduce the ARR, the patient may be a false negative, It is necessary to stop the drug for re-examination or further carry out the original aldehyde confirmation test; when the ARR test result is less than 30 negative results, if it is also detected that the patient contains drugs that can increase the ARR, it can be judged as negative; when the ARR When the test result is a positive result higher than 30, if the patient’s body contains drugs that can increase ARR, the patient may be a false positive, and the drug needs to be stopped for re-examination or further primary aldehyde confirmation test; when the ARR test result is When the positive result is higher than 30, if it is also detected that the patient contains drugs that can reduce the ARR, it can be judged as positive.

Further, the angiotensin I production rate is detected by detecting the angiotensin I concentration of the sample before incubation and the sample after incubation, according to the formula: angiotensin I production rate=(concentration of angiotensin I after incubation-before incubation Angiotensin Ⅰ concentration)/incubation time;

Further, the specific detection steps of the qualitative detection method for primary aldosteronism are:

(1) Sample pretreatment: 1) Take two 100 μL samples to be tested in parallel, add the generation buffer to the samples and mix well, one for incubation, and the other for the next step without incubation; 2) add Add the reaction stop solution to the sample that generates the buffer and mix well; 3) Add the protein precipitation agent containing internal standard to the sample that adds the reaction stop solution in step 2) and vortex and mix well; 4) Add the protein in step 3). The sample of the precipitant was mixed and centrifuged, and the supernatant was taken for detection on the machine;

(2) Use high-performance liquid chromatography tandem mass spectrometry system to detect angiotensin I in the solution to be detected obtained in the above steps and use unincubated samples to simultaneously screen 43 kinds of hypertension treatment drugs that affect ARR;

(3) For the samples whose screening results are positive according to the mass spectrometry parameters of therapeutic drugs in step (2), it is necessary to check the retention time of the standard substances of each hypertension treatment drug in the "screening drug mixed working solution" at the same time, and check the drug screening results. Whether the result is a false positive, to ensure the accuracy of the positive result of drug screening;

The protein precipitating agent containing internal standard is: using methanol, zinc sulfate aqueous solution and angiotensin I isotope internal standard to prepare a protein precipitating agent containing internal standard;

The generation buffer is: Tris and EDTA buffer containing PMSF;

The reaction termination solution is: formic acid or acetic acid;

The screening drug mixed working solution is: the screening drug mixed working solution prepared with the detection lower limit concentration with 43 standard substances of hypertension treatment drugs that affect ARR.

Further, the standard curve preparation: prepare the standard curve with different concentrations with the standard substance of angiotensin I;

At present, radioimmunoassay is generally used in the detection of ARR. Since radioimmunoassay cannot screen multiple therapeutic drugs through a single test, it is difficult to evaluate the effect of antihypertensive drugs on ARR if the patient does not stop taking drugs according to the regulations before ARR detection. detection impact.

The present invention has been proved by a large number of studies that when plasma renin activity (PRA) is detected by liquid chromatography tandem mass spectrometry, 43 kinds of hypertension treatment drugs that affect ARR can be detected synchronously, and a more accurate judgment of ARR can be realized in one step. And there is no need to stop the drug. A large number of clinical experiments have proved that the test results are accurate and reliable, and it is very worthy of large-scale clinical application.

In the process of quantitative detection of renin activity and synchronous detection of hypertension treatment drugs that affect ARR, the present invention uses a protein precipitation method to pretreat the sample, and simultaneously realizes the detection of angiotensin I and Effective extraction of antihypertensive drugs; then use the high-throughput characteristics of liquid chromatography tandem mass spectrometry technology to perform simultaneous detection of multiple indicators on the extracted samples, and detect the content of angiotensin I at the same time, forty-three treatments that affect ARR effective drug screening. The joint analysis of ARR detection value and interfering drug screening results can effectively reduce the false positive or false negative results of the detection while avoiding patients to stop drug treatment, and improve the accuracy of ARR judgment.

The detection method provided by the invention requires a small amount of samples, and 100 μL of plasma samples can detect the content of angiotensin I in the plasma before incubation and conduct qualitative screening for various antihypertensive drugs that affect the value of ARR.

The pretreatment of the detection method provided by the present invention only needs to obtain the solution to be tested through protein precipitation, without the need for complicated liquid-liquid extraction or SPE process, and avoids the reduction of the extraction rate of screening drugs with different characteristics caused by complex pretreatment. Realize high-throughput processing and detection of samples.

Further, the incubation refers to adding the sample to the generation buffer and mixing it evenly, and then putting it into a 37°C water bath for 3 hours; when detecting 43 kinds of hypertension treatment drugs that affect ARR, an unincubated sample is used; the mobile phase of the liquid phase A is 0.1% formic acid aqueous solution, mobile phase B is methanol solution with 0.1% formic acid by volume, and gradient elution is carried out.

Further, when the mass spectrometry is detected, a triple quadrupole mass spectrometer is used, the instrument model is SCIEX 4500MD, and the positive ion mode (ESI+) and the multiple reaction monitoring MRM mode of the electrospray ion source are used for mass spectrometry detection.

In the detection method provided by the invention, the liquid mass analysis time for each sample is only 3.8 minutes, and the detection efficiency is high.

For the drugs that are screened positive according to the mass spectrometry MRM parameters, compare them with the retention time of the drug standard in the "screening drug mixed working solution" to ensure the accuracy of the positive screening results.

On the other hand, the present invention provides a method for detecting renin activity, which mainly adopts the above-mentioned detection of angiotensin I production rate in plasma samples and qualitative screening of hypertension treatment drugs that affect ARR. The results were analyzed to determine the actual content of renin activity.

In another aspect, the present invention provides a method for detecting renin activity in plasma samples by liquid chromatography tandem mass spectrometry and synchronously detecting 43 kinds of hypertension drugs that affect ARR for clinical use in judging the ARR value and the actual content of renin activity.

Because any one of β-blockers, central α2 receptor agonists, and nonsteroidal anti-inflammatory drugs can reduce the renin activity of patients; potassium-sparing diuretics or potassium-sparing diuretics can make patients Any one of angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, and calcium ion antagonists can increase the renin activity of patients. Therefore, the method of detecting renin activity in plasma samples by liquid chromatography tandem mass spectrometry and synchronously detecting 43 kinds of hypertensive drugs that affect ARR can not only determine the ARR value qualitatively but also determine the actual value of renin activity while avoiding drug withdrawal. The content provides a more accurate judgment.

The present invention has the following beneficial effects:

(1) While performing renin activity (PRA) detection, realize the qualitative screening of 43 hypertension treatment drugs that affect the ARR value, and jointly analyze the ARR detection value combined with the interference drug screening results to prevent patients from stopping drug treatment At the same time, it effectively reduces the false positive or false negative results of ARR detection, improves the accuracy of actual clinical renin activity detection, and is convenient for clinical promotion and application.

(2) During the detection of renin activity, the protein precipitation method is used to pre-treat the sample. While the protein precipitation is carried out on the sample, the effective extraction of angiotensin I and 43 kinds of hypertension treatment drugs can be simultaneously realized , so as to perform synchronization detection. The solution to be tested can be obtained by pretreatment only through protein precipitation, without complex liquid-liquid extraction or SPE process, avoiding the reduction in the extraction rate of screening drugs with different characteristics caused by complex pretreatment.

(3) Using the high-throughput, high-specificity, and high-sensitivity characteristics of liquid chromatography tandem mass spectrometry technology, simultaneous detection of multiple indicators is performed on the extracted samples. While detecting the content of angiotensin I, the forty-three factors that affect ARR Effective screening for therapeutic drugs.

(4) The required sample size is small, 100 μL plasma sample can detect the content of angiotensin I in the plasma before incubation and conduct qualitative screening for 43 kinds of hypertension treatment drugs that affect the value of ARR.

(5) During the detection process, while the quantitative detection of angiotensin I is carried out, the MRM mass spectrometry parameters of the screening drug are used for the initial screening of the therapeutic drug. The retention time of each therapeutic drug in the drug is checked, and the positive detection results are reviewed to ensure the accuracy of the drug screening results.

(6) The liquid mass analysis time of each sample is only 3.8 minutes, and the detection efficiency is high.

(7) It can provide an efficient and accurate screening method for the screening of drugs for the treatment of primary aldolism, and provide a reliable basis for clinical diagnosis.

Description of drawings

Fig. 1 is the flow chart of sample detection in embodiment 1

Figure 2-13 is the detection spectrum of 43 kinds of hypertension drugs that affect ARR in Example 1

Fig. 14 is the detection spectrum of the aldosterone in embodiment 1

Fig. 15 is the plasma original aldehyde screening report sheet in embodiment 1

Detailed ways

The present invention will be described in further detail below with reference to the accompanying drawings and embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention, but do not limit it in any way. The reagents used in this example are all known products obtained by purchasing commercially available products.

Embodiment 1: the qualitative detection method of ARR provided by the present invention

1. Solution preparation:

Preparation of ZnSO ₄ solution: Weigh 3.56 g of ZnSO ₄ .7H ₂ O and dissolve it in 40 mL of water to prepare an 89 mg/mL ZnSO ₄ .7H ₂ O aqueous solution. The volume can be adjusted proportionally as needed and stored at room temperature.

Generation buffer: 12.11g of tris (TRIS) and 7.4g of ethylenediaminetetraacetic acid (EDTA) into a 100mL volumetric flask, add deionized water to 90mL, and sonicate for 30min until it dissolves evenly. Add deionized water to the mark and mix well. Transfer to a polypropylene storage container. Adjust the pH to 5.45-5.50 with acetic acid and store at -20°C. Prepared on the day of detection and analysis, 100 μL of 100 mM PMSF (0.174 g PMSF dissolved in 10 mL of methanol) solution was added to 10 mL of the generation buffer to prepare the generation buffer (pH 5.4-5.6).

Reaction termination solution: formic acid or acetic acid.

Protein precipitant containing internal standard: draw 50μL 1μg/mL internal standard stock solution of angiotensin I (methanol:water 1:1), and 100uL 89mg/mL zinc sulfate (ZnSO4.7H2O) aqueous solution, place in a 10mL centrifuge Add 9850 μL of methanol to the tube, and mix well to obtain the protein precipitant containing angiotensin I internal standard.

Screening drug mixed working solution: Dissolve 43 kinds of drug standard substances and some metabolite standard substances in DMSO to prepare the corresponding mother solution. The concentration of the screening drug is prepared as a mixed working solution of the detection limit concentration.

2. Sample testing:

The detection flow chart is shown in Figure 1.

(1) Reagent preparation: first add 20 μL of generation buffer to two clean 1.2 mL 96-well collection plates for subsequent sample pretreatment.

(2) Sample thawing: The plasma sample to be tested was thawed in ice water (0°C) until it melted.

(3) Sampling: Take two 100 μL plasma samples in parallel and transfer them to the two plates prepared in step (1), and test the unincubated and incubated samples respectively. The remaining samples are immediately frozen at -20°C. Screening was performed on unincubated samples).

(4) Treatment of incubated samples: seal a batch of samples in step (3) with a silica gel pad, vortex briefly, and then put them in a 37°C water bath for 3 hours. After 3 hours, add 12 μL reaction stop solution formic acid, vortex, Then add 200 μL internal standard working solution containing 5 ng/mL angiotensin I isotope internal standard ANG I- ¹³ C ¹⁵ N, vortex and mix well, then centrifuge at 15,000 rpm at 4°C for 10 minutes, take 100 μL of supernatant and add it into 96 wells. The sample is analyzed by liquid chromatography tandem mass spectrometer (a large number of research data prove that the incubation sample cannot be used in the drug screening process, and the test results are inaccurate or difficult to detect. The specific experimental data are omitted here. The incubation sample here is only for the calculation of renin activity. use).

(5) Treatment of unincubated samples: Seal another batch of samples remaining in step (3) with a sealing gasket, and then vortex and mix well. Immediately add 12 μL reaction stop solution formic acid, vortex, then add 200 μL internal standard working solution containing angiotensin I isotope internal standard, vortex and mix well, then centrifuge at 15000 rpm at 4 °C for 10 minutes, take 100 μL supernatant and add to 96 wells The sample is injected into the liquid chromatography tandem mass spectrometer for sample analysis (the unincubated sample of this step is used in the drug screening process, and the unincubated sample is not only used for renin activity calculation, but also for drug screening that interferes with ARR).

(6) Calculation of renin activity: renin activity is equal to the production rate of angiotensin I, and its unit is ng/mL/hr. The calculation formula is: (concentration of angiotensin I after incubation-concentration of angiotensin I before incubation) /incubation time.

(7) When performing liquid chromatography tandem mass spectrometry analysis, the liquid chromatography adopts gradient elution, and the reverse phase chromatography establishes the separation conditions of the analyte as follows: the chromatographic column is Phenomenex C18 (2.6 μm, 50*2.1mm), and the flow rate is 0.6mL/min, the column temperature is 40°C; the mobile phase A is an aqueous solution of formic acid with a volume ratio of 0.1%, and the mobile phase B is a methanol solution with a volume ratio of 0.1% formic acid. The volume ratio of mobile phase A and mobile phase B is 90-5%: 10-95%. The gradient program is shown in Table 1, and the retention times of angiotensin I and its isotope internal standard are respectively: 1.92min.

Table 1. Gradient elution program

time (min) Flow rate (mL/min) Mobile phase A(%) Mobile phase B(%) 0 0.6 90 10 1 0.6 90 10 2 0.6 5 95 3.2 0.6 5 95 3.25 0.6 90 10 3.8 0.6 90 10

The retention times of the standard substances of each hypertension treatment drug in the "screening drug mixed working solution" are shown in Table 2:

Table 2, the retention time of the standard substance of hypertension treatment drug

For mass spectrometry detection, a triple quadrupole mass spectrometer was used for quantitative detection of angiotensin I before and after incubation and qualitative screening of antihypertensive drugs. The reaction monitoring MRM mode is used for mass spectrometry detection, and the corresponding mass spectrometry detection method settings are shown in Table 3 and Table 4:

Table 3. Mass spectrometry detection parameter settings

Table 4. Mass spectrometry parameters of each analyte

The detection maps of 43 hypertension drugs that affect ARR are shown in Figure 2-13, in which Figure 2 shows the potassium-sparing diuretic Amiloride, the CCB drug Amilodipine, and the beta-blocker The detection spectra of Arotinolol and the non-steroidal anti-inflammatory drug Aspirin; Figure 3 shows the β-blocker Atenolol and the ACEI drug Benazepril , CCB drug Benidipine (Benidipine) and β-receptor blocker Besoprolol (Bisoprolol) detection spectrogram; Candesartan), ACEI drug Captopril (Captopril) and ACEI drug Enalapril (Enalapril); The detection spectra of Esmolol, CCB drug Felodipine (Felodipine) and CCB drug flunarizine (Flunarizine); Furosemide), the potassium-sparing diuretic Hydrochlorothiazide (Hydrochlorothiazide) and the ACEI drug Imidapril (Imidapril); Figure 7 is the potassium-sparing diuretic Indapamide (Indapamide), the ARB drug Irbesartan (Irbesartan) , β receptor blocker Labetolol (Labetolol) and CCB drug Lercanidipine (Lercanidipine); Figure 8 shows the ACEI drug Lisinopril (Lisinopril), ARB drug Losartan (Losartan), The detection spectrum of the central α2 receptor agonist methyldopa (Methyldopa) and the β receptor blocker Metoprolol (Metoprolol); Figure 9 is the β receptor blocker Nebivolol (Nebivolol), CCB The detection spectra of drug nifedipine metabolite (Nifedipine), CCB drug nifedipine (Nifedipine) and CCB drug nicardipine (Nicardipine); Figure 10 is ARB drug olmesartan (Olmesartan), CCB drug nimodipine ( Nimodipine), β receptor blocker propranolol (Propranolol) and ACEI drug perindopril (Perindopril) detection spectrogram; Fig. 11 is ACEI drug ramipril (Ramipril), ACEI drug quinapril (Quinapril), β receptor blocker Sotalol (Sotalol) and potassium-sparing diuretic Spironolactone (Spironolactone); Tan (Telmisartan), potassium excretion diuretic Torasemide (Torasemide) and ACEI drug trandolapril (Trandolapril) detection spectrum; Figure 13 is ARB drug Valsartan (Valsartan), angiotensin I (Ang I ) and the detection spectrum of angiotensin I isotope internal standard (Ang I 13C15N). Among them, the X and Y axis coordinates of the detection spectrum of 43 kinds of hypertension drugs affecting ARR are consistent with the X and Y axis coordinates of the detection spectrum of the potassium-sparing diuretic drug Amiloride in FIG. 2 .

(8) Detection of aldosterone: The sample pretreatment process for the detection of aldosterone in plasma is extracted by carrier liquid-liquid extraction and detected by liquid chromatography tandem mass spectrometry. The specific steps are as follows: pipette 300-450uL plasma, add 50uL aldosterone The standard solution was loaded onto the SLE plate, allowed to stand for ten minutes, and eluted with 1.5 mL of a mixed solvent of ethyl acetate and n-hexane (1:1). After the eluent was dried, the sample was reconstituted and detected by liquid chromatography tandem mass spectrometer, using ESI+ detection mode, the quantitative MRM ion pair of aldosterone Aldosterone was 361.3/315.1; the MRM ion pair of aldosterone internal standard Aldosterone-d8 was 369.4/323.1 ; The representative detection spectrogram is shown in Figure 14. A large number of studies have proved that the pretreatment and detection process of aldosterone cannot simultaneously screen 43 drugs that affect ARR, and the detection results are inaccurate or difficult to detect. The reason may be that the pretreatment process is not suitable for the extraction of 43 drugs that affect ARR. The experimental data are omitted.

(9) Drug screening test report: In the plasma primary aldehyde screening report (as shown in Figure 15), summarize the screening results of drug factors that lead to ARR false positive or false negative, clinical combined ARR value and drug screening results , which can effectively exclude some false positive or false negative results caused by drug interference.

Example 2: False negative clinical sample detection verification

In this example, clinical samples were detected and verified, and the detection method provided in Example 1 was used to detect and qualitatively judge ARR. The detection results of clinical samples are shown in Table 5.

Table 5. Test results of clinical samples

According to the above test results of the sample, the ARR is less than the cut-off value (30) for disease diagnosis, therefore, the patient of this sample should be judged as negative. However, combined with the screening results of antihypertensive drugs in the patient in Table 6, ARB and CCB drug tests are positive, both of which can lead to false negatives in ARR detection. Comprehensive evaluation, although the ARR result of this patient sample is lower than the cut-off value, it is still possible For positive patients, further confirmatory tests are recommended.

Table 6. Drug screening results of patients

drug factor screening results drug name Effects on Aldosterone Effects on Renin Impact on ARR beta-blockers - none ↓ ↓↑ ↑ (false positive) Central α2 receptor blockers - none ↓ ↓↓ ↑ (false positive) NSAIDs - none ↓ ↓↓ ↑ (false positive) Potassium-sparing diuretics - none →↑ ↑↑ ↓(false negative) Potassium-sparing diuretics - none ↑ ↑↑ ↓(false negative) ACEI - none ↓ ↑↑ ↓(false negative) ARBs + Valsartan ↓ ↑↑ ↓(false negative) Dihydropyridine CCBs + Amlodipine →↓ ↑ ↓(false negative)

Confirmed by the captopril confirmatory test, the patient was indeed a positive patient with primary aldosteronism.

Example 3: False Positive Clinical Sample Detection Verification

In this example, clinical samples were detected and verified, and the detection method provided in Example 1 was used to detect and qualitatively judge ARR. The detection results of clinical samples are shown in Table 7.

Table 7. Test results of clinical samples

According to the above test results of the sample, the ARR is greater than the cut-off value (30) for disease diagnosis, therefore, the patient of this sample should be judged as positive. However, combined with the screening results of antihypertensive drugs in patients in Table 8, the positive results of β-blockers and central α2 receptor agonists, both of which can lead to false positives in ARR detection, comprehensive evaluation, although this patient sample If the ARR result is higher than the cut-off point, it may still be negative, and further confirmation tests are recommended.

Table 8. Drug screening results of patients

drug factor screening results drug name Effects on Aldosterone Effects on Renin Impact on ARR beta-blockers + Metoprolol ↓ ↓↓ ↑ (false positive) Central α2 receptor blockers + Methyldopa ↓ ↓↓ ↑ (false positive) NSAIDs - - ↓ ↓↓ ↑ (false positive) Potassium-sparing diuretics - - →↑ ↑↑ ↓(false negative) Potassium-sparing diuretics - - ↑ ↑↑ ↓(false negative) ACEI - - ↓ ↑↑ ↓(false negative) ARBs - - ↓ ↑↑ ↓(false negative) Dihydropyridine CCBs - - →↓ ↑ ↓(false negative)

Confirmed by the captopril test, the patient was indeed a negative patient.

Example 4: Positive clinical sample detection verification

In this example, clinical samples were tested and verified, and the detection method provided in Example 1 was used to detect and qualitatively judge ARR. The test results of clinical samples are shown in Table 9.

Table 9. Test results of clinical samples

According to the above test results of the sample, the ARR is greater than the cut-off value (30) for disease diagnosis, therefore, the patient of this sample should be judged as positive. At the same time, combined with the screening results of antihypertensive drugs in the patient in Table 10, ARB and potassium-sparing diuretic drugs are positive, both of which can lead to false negatives in ARR detection. Comprehensive evaluation, this patient sample is taking ARBs that can cause false negatives And potassium excretion diuretic drugs, if the ARR result is still higher than the cut-off value, the patient may be a positive sample, and the diagnosis test can be carried out without stopping the drug.

Table 10. Drug screening results of patients

drug factor screening results drug name Effects on Aldosterone Effects on Renin Impact on ARR beta-blockers - - ↓ ↓↓ ↑ (false positive) Central α2 receptor blockers - - ↓ ↓↓ ↑ (false positive) NSAIDs - - ↓ ↓↓ ↑ (false positive) Potassium-sparing diuretics + Hydrochlorothiazide →↑ ↑↑ ↓(false negative) Potassium-sparing diuretics - - ↑ ↑↑ ↓(false negative) ACEI - - ↓ ↑↑ ↓(false negative) ARBs + Candesartan ↓ ↑↑ ↓(false negative) Dihydropyridine CCBs - - →↓ ↑ ↓(false negative)

Although the present invention is disclosed above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, so the protection scope of the present invention should be based on the scope defined in the claims.

Claims (6)

1. The synchronous qualitative detection method for the antihypertensive drugs affecting ARR in the process of detecting the activity of renin by a liquid chromatography-tandem mass spectrometry method is characterized in that the method can be used for judging and analyzing the value of ARR by combining the concentration of aldosterone by detecting the generation rate of angiotensin I in a plasma sample and the concentration of the antihypertensive drugs affecting ARR, and analyzing and judging the ARR to be one of negative, positive, false negative or false positive; the total of 43 hypertension treatment medicines affecting ARR are respectively:

nine beta-blockers include alolol, atenolol, bisoprolol, esmolol, labetalol, metoprolol, nebivolol, propranolol, sotalol;

five kinds of potassium-expelling diuretics include bumetanide, furosemide, hydrochlorothiazide, indapamide and torsemide;

the three potassium-retaining diuretics include amiloride, eplerenone and spironolactone;

ten angiotensin converting enzyme inhibitors include benazepril, captopril, enalapril, fosinopril, imidapril, lisinopril, perindopril, quinapril, ramipril, trandolapril;

six angiotensin receptor antagonists include candesartan, irbesartan, losartan, olmesartan, telmisartan, valsartan;

eight calcium antagonists include amlodipine, benidipine, felodipine, flunarizine, lercanidipine, nicardipine, nifedipine, nimodipine;

a central α2 receptor agonist comprises methyldopa;

a non-steroidal anti-inflammatory drug comprising aspirin; the ARR has a calculation formula as follows: ARR = aldosterone concentration/angiotensin i production rate;

the beta-receptor blocker, the central alpha 2 receptor agonist and the nonsteroidal anti-inflammatory drug can all cause ARR to rise and cause false positive results to appear; the potassium-expelling diuretic, potassium-preserving diuretic, angiotensin converting enzyme inhibitor, angiotensin receptor antagonist and calcium ion antagonist can reduce ARR, and false negative result appears;

the step of analyzing the value of ARR is: analyzing and judging ARR as one of negative, positive, false negative or false positive through a clinical medication AI guiding system; the clinical medication AI guiding system comprises a patient information module, a database module and a decision tree system;

the analysis and judgment method of the decision tree system comprises the following steps: when the detection result of the ARR is less than 30, if the patient contains the drug capable of reducing the ARR, the patient can be judged to be false negative, and further diagnosis experiments or drug withdrawal checks are required; when the ARR detection result is less than 30, if the patient contains the medicine capable of raising the ARR, the patient can be judged as negative; when the detection result of the ARR is more than 30, if the medicine capable of increasing the ARR is detected in the patient at the same time, the patient can be judged to be false positive, and further diagnosis experiment or medicine stopping check is needed; when the ARR detection result is more than 30, if the patient contains the drug capable of reducing the ARR, the patient can be judged to be positive;

the non-incubated samples were used for detection of 43 hypertensive therapeutic agents affecting ARR; the mobile phase A of the liquid phase is 0.1% formic acid aqueous solution, the mobile phase B of the liquid phase is 0.1% formic acid methanol solution by volume ratio, the gradient procedure is shown in the following table,

the detection of the aldosterone adopts a carrier liquid-liquid extraction method for extraction, and adopts a liquid chromatography-tandem mass spectrometry method for detection.

2. The method of claim 1, wherein the information module is configured to record basic information of the patient; the database module is used for storing the detection result of ARR and the detection result of the hypertension treatment drug; and the decision tree system automatically analyzes and judges the ARR index as one of negative, positive, false negative or false positive according to the detection result.

3. The method of claim 2, wherein the rate of angiotensin i production is determined by measuring the concentration of angiotensin i in the pre-incubation sample and the post-incubation sample according to the formula: angiotensin i production rate = (angiotensin i concentration after incubation-angiotensin i concentration before incubation)/incubation time.

4. The method according to claim 3, wherein the specific detecting steps are as follows:

(1) Sample pretreatment: 1) Taking two 100mL samples to be tested in parallel, respectively adding a generating buffer solution into the samples, uniformly mixing, incubating one sample, and directly carrying out the next operation without incubating the other sample; 2) Adding a reaction stopping solution into the sample added with the generated buffer solution and uniformly mixing; 3) Adding an internal standard-containing protein precipitant into the sample added with the reaction stopping solution in the step 2) and uniformly mixing by vortex; 4) Uniformly mixing the sample added with the protein precipitant in the step 3), centrifuging, and taking supernatant for machine detection;

(2) Detecting angiotensin I and screening 43 antihypertensive drugs affecting ARR by adopting a high performance liquid chromatography tandem mass spectrometry system to the solution to be detected of the sample before incubation;

(3) For the sample with the positive drug according to the mass spectrum parameter screening result in the step (2), the retention time of the standard substance of each hypertension therapeutic drug in the screening drug mixed working solution is required to be simultaneously compared, whether the drug screening result is false positive or not is checked, and the accuracy of the drug screening positive result is ensured from two dimensions of the mass spectrum parameter and the retention time;

the protein precipitant containing the internal standard is as follows: methanol, zinc sulfate aqueous solution and an angiotensin I isotope internal standard are prepared into a protein sedimentation agent containing the internal standard;

the generating buffer solution is as follows: tris and EDTA buffers containing the angiotensin converting enzyme inhibitor PMSF (phenylmethylsulfonyl fluoride);

the reaction termination liquid is as follows: formic acid or acetic acid;

the screening medicine mixed working solution comprises the following components: the mixed working solution of the screening medicaments for detecting the lower limit concentration is prepared by using 43 standard substances of the hypertension therapeutic medicaments affecting the ARR.

5. The method according to claim 4, wherein the incubation is performed by adding the sample to the buffer and mixing the sample with the buffer, and then placing the sample in a water bath at 37 ℃ for 3 hours;

the retention time of the standard substance of each hypertension treatment drug in the screening drug mixed working solution is shown in the following table:

。

6. the method of claim 5, wherein the mass spectrometry is performed using a triple quadrupole mass spectrometer having a model SCIEX 4500MD, using positive ion mode (esi+) of electrospray ion source and multi-reaction monitoring MRM mode, and the corresponding mass spectrometry is set forth in the following table:

the mass spectrum parameters of each test object are shown in the following table:

。